DESCRIPTION: To continue an investigation into the mechanisms of translational control, the applicant proposes to focus on an ORF upstream of the HCMV UL4 gene which specifies a viral glycoprotein (gpUL4). The second ORF (uORF2) upstream of gpUL4 inhibits translation in both mammalian and yeast cells. During the last funding period, the applicant published five peer-reviewed articles, one in press article, and two review articles. The peer-reviewed publications were in strong basic science journals and represent primarily observations of translational regulation in transiently transfected cells or in in vitro translation systems. The applicant responded to a previous critique of this application by eliminating an analysis of a more complicated viral latency transcript with short upstream ORFs. Studies on the binding of uORF2 peptide to purified ribosomes was also eliminated. The applicant will now emphasize the construction of recombinant virus to test this phenomena in the context of the viral genome. Between funding periods the applicant has made the following progress: (i) the uORF2 peptide made in cell-free translation extracts was demonstrated to accumulate on ribosomes and remain linked to tRNA for the terminal proline codon. (ii) Mutation of the coding sequence at the carboxyl end alleviated inhibition of downstream translation and the uORF2-peptidyl-tRNA was efficiently released. To continue this research the applicant proposes the following specific aims: (i) To demonstrate translational regulation by uORF2 in the context of the HCMV genome, point mutations in the uORF2 will be recombined into the virus. (ii) To determine the mechanisms of translational regulation at the uORF2, the RNA and protein factors involved in the termination of translation will be investigated. In the first specific aim, the applicant proposes to introduce point mutations in the uORF2 and recombine these sequences into the virus using a set of overlapping genomic cosmids. The mutations will be in the AUG codon and in the terminal proline codon. The effect of the mutations in the context of the viral genome will be determined by measuring the gpUL4 in mRNA and protein. The extent of ribosomal stalling with wild type and mutant transcripts will be determined by a toe print assay. If the uORF2 peptide or the uORF2 peptide linked to tRNA is made in the HCMV infected cell, antibody to the uORF2 will be used for immunoprecipitation. The applicant also proposes to isolate cell lines for gpUL4 expression and determine their effect on viral replication. To investigate the long delay between appearance of the gpUL4 mRNA (8h) and the viral glycoprotein (48h), the applicant will substitute gpUL4 with an indicator gene that specifies the secreted embryonic alkaline phosphatase (SEAP) or follow the authentic gpUL4 with an antibody. Because of problems with obtaining good antibodies to gpUL4, the authentic gpUL4 would be his-tagged and epitope-tagged. Changes in the ribosome fractions at various times after infection would be measured at the 5' end of uORF2 and the 3' end of gpUL4 using an antisense oligonucleotide and RNase H to cleave the transcript and then measuring the cleavage products by RNase protection assay. In the second specific aim, the applicant proposes to elucidate the mechanism of translational regulation by the uORF2. Transient transfection studies in mammalian cells indicated uORF2 downregulated expression of a reported gene. Preliminary data by the applicant suggest that downregulation of translation by uORF2 also works in S. cerevisiae, but mutations in the uORF2 (P22A and K10Q/S12P) alleviate the down regulation. Ribosome stalling in the yeast system will also be assayed by the toe print assay and by immunoprecipitation. The applicant proposes to extend this work by using a yeast genetic approach with selectable markers and indicator gene expression. Yeast genes that may have a role in overcoming the uORF2 block would be investigated. tRNA suppressers for the uORF2 termination codon would be of interest. Cell free extracts would be used to investigate eukaryotic release factor (eRF1 and eRF3) and their association with ribosomes and the uORF2. The kinetics of translation termination would be measured by the "toe print decay assay". Lastly, mammalian cell transfection studies with plasmids expressing eRF1, eRF3, and uORF2-beta-gal would be used to test the effects of wild type and mutant sequences in the uORF2.